This proposal studies the role of hemolysins as virulence factors for Bacillus anthracis. Hemolysins allow certain virulent bacterial species to escape the phagosome of macrophages. Using the sequence of Listeria monocytogenes hemolysins for comparison, the incomplete genome of B. anthracis was screened for the presence of hemolysin genes. Five open reading frames containing protein-coding sequences homologous to Listeria hemolysins or associated proteins were discovered in the B. anthracis chromosome. These hemolytic proteins, named anthralysins 0, A, B (AnLO, AnLA, AnLB); p3058, and p3201, have not been previously characterized. The goals of this proposal are to: (1) Characterize these hemolytic genes by studying gene expression patterns and regulation; (2) Address the role of each individual anthralysin gene in anthrax pathogenesis; and (3) Study means to inhibit the activity of anthralysins. Such understanding could lead to the development of new antibiotic compounds which act by inhibiting hemolytic proteins and preventing release of anthrax bacilli from the macrophage phagosome, preventing anthrax-associated macrophage death and blocking the infection before it can become systemic.